identification-footprints-rnaprotein-complexes-via-rna

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing (RIPiT-Seq)

RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT ...

The Ohio State University

Throughout their lifetimes, messenger RNAs (mRNAs) associate with proteins to form ribonucleoproteins (mRNPs). Since the discovery of the first mRNP component more than 40 years ago, what is known as the mRNA interactome now comprises >1,000 proteins. These proteins bind mRNAs in myriad ways with varying affinities and stoichiometries, with many assembling onto nascent RNAs in a highly ordered process during transcription and precursor mRNA (pre-mRNA) processing. The nonrandom distribution of major mRNP proteins observed in transcriptome-wide studies leads us to propose that mRNPs are organized into three major domains loosely corresponding to 5' untranslated regions (UTRs), open reading frames, and 3' UTRs. Moving from the nucleus to the cytoplasm, mRNPs undergo extensive remodeling as they are first acted upon by the nuclear pore complex and then by the ribosome. When not being actively translated, cytoplasmic mRNPs can assemble into large multi-mRNP assemblies or be permanently disassembled and degraded. In this review, we aim to give the reader a thorough understanding of past and current eukaryotic mRNP research. 

The Clothes Make the mRNA: Past and Present Trends in mRNP Fashion.

Introns are found in 5' untranslated regions (5'UTRs) for 35% of all human transcripts. These 5'UTR introns are not randomly distributed: Genes that encode secreted, membrane-bound and mitochondrial proteins are less likely to have them. Curiously, transcripts lacking 5'UTR introns tend to harbor specific RNA sequence elements in their early coding regions. To model and understand the connection between coding-region sequence and 5'UTR intron status, we developed a classifier that can predict 5'UTR intron status with >80% accuracy using only sequence features in the early coding region. Thus, the classifier identifies transcripts with ' proximal-ntron-inus-like-coding regions ("5IM" transcripts). Unexpectedly, we found that the early coding sequence features defining 5IM transcripts are widespread, appearing in 21% of all human RefSeq transcripts. The 5IM class of transcripts is enriched for non-AUG start codons, more extensive secondary structure both preceding the start codon and near the 5' cap, greater dependence on eIF4E for translation, and association with ER-proximal ribosomes. 5IM transcripts are bound by the exon junction complex (EJC) at noncanonical 5' proximal positions. Finally, -methyladenosines are specifically enriched in the early coding regions of 5IM transcripts. Taken together, our analyses point to the existence of a distinct 5IM class comprising ∼20% of human transcripts. This class is defined by depletion of 5' proximal introns, presence of specific RNA sequence features associated with low translation efficiency, -methyladenosines in the early coding region, and enrichment for noncanonical binding by the EJC.

A common class of transcripts with 5'-intron depletion, distinct early coding sequence features, and -methyladenosine modification.

During messenger RNA (mRNA) biogenesis and processing in the nucleus, many proteins are imprinted on mRNAs assembling them into messenger ribonucleoproteins (mRNPs). Some of these proteins remain stably bound within mRNPs and have a long-lasting impact on their fate. One of the best-studied examples is the exon junction complex (EJC), a multiprotein complex deposited primarily 24 nucleotides upstream of exon-exon junctions as a consequence of pre-mRNA splicing. The EJC maintains a stable, sequence-independent, hold on the mRNA until its removal during translation in the cytoplasm. Acting as a molecular shepherd, the EJC travels with mRNA across the cellular landscape coupling pre-mRNA splicing to downstream, posttranscriptional processes such as mRNA export, mRNA localization, translation, and nonsense-mediated mRNA decay (NMD). In this review, we discuss our current understanding of the EJC's functions during these processes, and expound its newly discovered functions (e.g., pre-mRNA splicing). Another focal point is the recently unveiled in vivo EJC interactome, which has shed new light on the EJC's location on the spliced RNAs and its intimate relationship with other mRNP components. We summarize new strides being made in connecting the EJC's molecular function with phenotypes, informed by studies of human disorders and model organisms. The progress toward understanding EJC functions has revealed, in its wake, even more questions, which are discussed throughout. WIREs RNA 2017, 8:e1411. doi: 10.1002/wrna.1411 For further resources related to this article, please visit the WIREs website.

The exon junction complex: a lifelong guardian of mRNA fate.

Argonaute proteins (AGOs) are loaded with small RNAs as guides to recognize target mRNAs. Since the target specificity heavily depends on the base complementarity between two strands, it is important to identify small guide and long target RNAs bound to AGOs. For this purpose, next-generation sequencing (NGS) technologies have extended our appreciation truly to the nucleotide level. However, the identification of RNAs via NGS from scarce RNA samples remains a challenge. Further, most commercial and published methods are compatible with either small RNAs or long RNAs, but are not equally applicable to both. Therefore, a single method that yields quantitative, bias-free NGS libraries to identify small and long RNAs from low levels of input will be of wide interest. Here, we introduce such a procedure that is based on several modifications of two published protocols and allows robust, sensitive, and reproducible cloning and sequencing of small amounts of RNAs of variable lengths. The method was applied to the identification of small RNAs bound to a purified eukaryotic AGO. Following ligation of a DNA adapter to RNA 3'-end, the key feature of this method is to use the adapter for priming reverse transcription (RT) wherein biotinylated deoxyribonucleotides specifically incorporated into the extended complementary DNA. Such RT products are enriched on streptavidin beads, circularized while immobilized on beads and directly used for PCR amplification. We provide a stepwise guide to generate RNA-Seq libraries, their purification, quantification, validation, and preparation for next-generation sequencing. We also provide basic steps in post-NGS data analyses using Galaxy, an open-source, web-based platform.

Cloning and Identification of Recombinant Argonaute-Bound Small RNAs Using Next-Generation Sequencing.

The exon junction complex (EJC) deposited upstream of mRNA exon junctions shapes structure, composition, and fate of spliced mRNA ribonucleoprotein particles (mRNPs). To achieve this, the EJC core nucleates assembly of a dynamic shell of peripheral proteins that function in diverse post-transcriptional processes. To illuminate consequences of EJC composition change, we purified EJCs from human cells via peripheral proteins RNPS1 and CASC3. We show that the EJC originates as an SR-rich mega-dalton-sized RNP that contains RNPS1 but lacks CASC3. Sometime before or during translation, the EJC undergoes compositional and structural remodeling into an SR-devoid monomeric complex that contains CASC3. Surprisingly, RNPS1 is important for nonsense-mediated mRNA decay (NMD) in general, whereas CASC3 is needed for NMD of only select mRNAs. The switch to CASC3-EJC slows down NMD. Overall, the EJC compositional switch dramatically alters mRNP structure and specifies two distinct phases of EJC-dependent NMD.

Guramrit Singh

Department of Molecular Genetics,
Center for RNA Biology,
Department of Molecular Genetics, Center for RNA Biology

The Ohio State University

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing (RIPiT-Seq)

Guramrit Singh

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Molecular Genetics,Center for RNA Biology,Department of Molecular Genetics, Center for RNA Biology

The Ohio State University

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing (RIPiT-Seq)

Department of Molecular Genetics,
Center for RNA Biology,
Department of Molecular Genetics, Center for RNA Biology